Previous investigations have been directed at developing methods for identifying specific peptide-peptide or peptide-nucleotide interactions.
U.S. Pat. No. 5,498,530 to Schatz and U.S. Pat. No. 5,270,170 to Schatz describe a method for isolating novel peptide ligands that bind to receptor molecules of interest using phage display combinatorial library techniques. The '530 patent concerns the method of constructing the combinatorial peptide library used in the '170 patent. The methods described involve the use of combinatorial libraries to express fusion proteins, but employ in vitro binding affinity. These references involve neither a reporter plasmid having a reporter gene driven by a target DNA sequence nor a transactivating domain of a transcription factor in the fusion protein. They do not teach identification of peptides by functional expression and selection.
Rebar and Pabo, Science 263, 671-673 (1994) discloses a method for identifying peptides that have high affinity to specific nucleotide sequences, specifically zinc finger binding sequences, using phage display combinatorial library techniques and affinity selection. Like Schatz, the method disclosed here, while employing fusion protein expression and random oligonucleotides cloned into a DNA vector, does not involve a functional expression selection strategy.
PCT Application WO 96/06166 to Choo et al., and Choo et al., Nature 372, 642-645 (1994), disclose a method for identifying zinc-finger DNA-binding proteins using phage display combinatorial library techniques and affinity selection in combination with rational design. In addition to the method of identification, the patent also describes a method of using the selected DNA-binding peptide to block/alter gene expression in mammalian cells. However, neither the paper nor the patent application discloses selection of DNA-binding peptides or drugs by functional expression.
U.S. Pat. No. 5,498,538 to Kay and Fowlkes describes a method for identifying a protein, polypeptide or peptide which binds to a ligand of choice by screening a library of recombinant vectors in which each vector expresses a fusion protein containing a binding domain and an effector domain that enhances expression or detection of the binding domain. The specification discloses only affinity selection using magnetic beads in vitro. Functional selection in vivo by activating the expression of a reporter gene in a reporter plasmid is not contemplated, and screening based on the death or survival of transformed cells is not envisioned.
The foregoing all fail to provide a method of identifying biologically significant peptide-DNA binding events. Accordingly, there is an ongoing need for improved methods of identifying and characterizing peptide-DNA interactions.